Conservative whole blood sample preparation technique

ABSTRACT

The method of this invention is directed to the rapid preparation of a whole blood sample for photooptical analysis. In the preferred embodiments of this method, a whole blood sample, lytic reagent system and immunological stain (optional) are contacted with the sample in a common reaction vessel (i.e. cuvette or test tube), with gentle asymmetric vortex mixing, so as to maintain the particulate matter of the sample at an essentially homogeneous concentration throughout the sample. An aliquot of the contents of the reaction vessel can, thereafter, be analyzed for identification and/or quantification of the analyte of interest. This process is conservative of the various endogenous constituents of the sample since virtually all of the manipulative steps involved in the preparation of the sample occur within a common vessel and any excess (unconsumed or unreacted) reagents and stain need not be separated from the sample during such preparation, thus, avoiding the multiple wash steps traditionally associated with this process. This process is uniquely applicable to the conservative preparation of aged whole blood samples (samples in which the endogenous nutrients have been essentially completely consumed) and whole blood samples from diseased state patients wherein the disease is manifest by changes in the morphology, total number and/or relative concentration of one or more of the lymphocyte subpopulations (i.e. B and/or T-cells).

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a method. More specifically, thisinvention concerns a unique process for the rapid preparation ofparticulate analytes, notably cells, cell organelles and the like, foranalysis by photooptical means. This process is particularly well suitedfor analysis of cellular analytes which are present in smallconcentrations and/or where a change in their relative proportion toanother cell population is diagnostically significant. The ability ofthis process to afford such advantage over the more traditionaltechniques is based upon the conservative nature of the preparationprocess. The term "conservative" as used herein is intended asdescriptive of the ability of this process to preserve virtually all ofthe blood components, with the exception of erythrocytes, for lateranalysis. The integrity of the sample, with respect to the conservedcomponents, also provides the first truly reliable means for theestablishment of standards against which other samples can be measured.This process is particularly well adapted to analysis of aged samples ofwhole blood, and to blood samples from disease state patients, whereincell integrityis difficult to maintain and thus an accurate analysisheretofore impossible. This process is particularly well suited forrapid preparation of whole blood samples for analysis by flow cytometry.

2. Description of the Prior Art

The traditional techniques for the isolation and staining of theleukocyte fraction of whole blood has typically involved numerousphysical manipulations of the sample and an inordinate number of washsteps. As has been appreciated and reported in the technical literature,these traditional techniques inherently result in some finite depletionof the leukocyte population from the sample; at least some alteration inthe gross morphology of the leukocyte population which remain in thesample; at least some alteration in the markers on the surface of theleukocyte population; and, at least some displacement of the strain onthe surface of the cell population of interest.

The techniques which have been used up to now in the preparation ofleukocytes from whole blood samples for subsequent analysis havetraditionally involved: (1) the preparation of a buffy coat fraction bycontrolled centrifugation of the whole blood sample, followed bystaining with a fluorochrome labelled conjugate; (2) concurrenttreatment of a whole blood sample with a lytic reagent and afluorochrome labelled conjugate; or (3) the preparation of a buffy coatfraction by controlled centrifugation of the whole blood sample andthereafter subjecting the buffy coat sample to further enrichment byFicoll-Paque density gradient centrifugation. Such enrichment permitsthe recovery of an interfacial layer containing enriched mononuclearcells which are thereafter stained with a fluorochrome labelledconjugate.

Each of the above preparative procedures is labor intensive, requiresrepeated physical manipulation of the fraction containing the leukocytesof interest and is time consuming. Where conventional centrifugationtechniques are used to obtain an enriched leukocyte sample, cell losswill invariably take place and, thus accurate determination of therelative concentration of the cellular population of interest isvirtually impossible. The shortcomings in the above procedures have beenrecognized for some time and at least one alterative has been proposedin the technical literature, articles by Caldwell, appearing inA.J.C.P., vol. 88:4 (1987) pp. 447-456; and et al, A.J.C.P., Vol. 86:5(1986), pp. 600-607. The Caldwell articles, however, focus only upon onefacet of this problem, which he believes to be the principle detractorfrom prior procedures. More specifically, Caldwell emphasizes thatexcessive washing of the leukocytes samples subsequent to staining andprior to analysis can introduce analytical error into the analysis,especially where leukocytes are observed in certain classes of diseasedpatients. Caldwell suggests adoption of a "no wash" technique to reduceprocessing time and analytical error which is inherent in the extensivemanipulation treatment of the leukocyte sample prior to its ultimateanalysis. The "no wash" technique proposed by Caldwell involvesretention of the traditional techniques for preparation of an enrichedlymphocyte fraction (i.e., Ficoll-Paque density gradient centrifugation,which itself contemplates multiple wash steps for removal of theFicoll-Paque reagents). The enriched sample of lymphocytes is,thereafter, stained with a fluorochrome dye conjugated to a monoclonalantibody. Caldwell's improvement resides in the observation of enhancedfluorescence intensity in both background and positive peaks of ahistogram when the unbound conjugate is allowed to remain in the sample.Caldwell attributes this observation of enhanced fluorescence intensityto his elimination of the wash steps after staining. He hypothesizesthat the elimination of such wash step is less disruptive upon thestained lymphocyte fraction, thus, enabling preservation of theimmunochemical bond between the conjugate and the surface markers on thecell population of interest. As is clearly evident from review ofCaldwell's "no wash" procedure, the repeated trauma of mixing, vortexingand centrifugation is only somewhat reduced.

The "enrichment" of the leukocyte sample through the use of lyticreagents has not, up to now, proven to be a viable alternative to themore traditional centrifugation/density gradient techniques forpartitioning the whole blood sample into its various fractions. Thereason for this limitation is the inablity of prior lytic reagents toeffect hemolysis of the erythrocyte fraction of whole blood without alsotraumatizing the leukocyte fraction of the sample. These lytic reagentstypically cause both gross and subtle morphological changes in theleukocyte fraction and alternation in the surface markers on such cells.In the limited number of instances where lytic reagents have been usedwith some measure of success, additional reagents were required to beadded to the sample to protect the leukocytes from lysis by the lyticreagent, see for example, U.S. Pat. No. 4,637,986 (to Brown, et al). Asis evident from the review of the Brown patent, the physiologicalenvironment of the sample is modified dramatically by the addition ofboth lytic reagent and the solutes which are used to protect theleukocytes from lysis by the lytic reagent. The samples prepared inaccordance with Brown's reagents and technique can be subjected toanalysis by flow cytometry where differation of the three principlesub-populations is accomplished by conventional light scatteringmeasurements. Because of the alteration in the physilogical environmentof the sample, the integrity and immunochemical response of the surfacemarkers on the leukocytes of the samples is altered as well. Thus,refinement in the analysis of the leukocyte fraction by immunochemicaltechniques is precluded. Moreover, where the leukocyte fraction is froma sample which is "aged" (not fresh) or a diseased patient sample, thesensitivity of the leukocytes to such harsh treatment is increaseddramatically, thus, further limiting the usefulness of the Brownreagents and technique.

As is evident from the above discussion, both the physical and chemicaltrauma which can be effected upon the leukocyte fraction by the aboveprocedures, is likely to damage a number of the cells within thisfraction. Even more unfortunate, these procedures are more destructiveof the leukocytes (i.e. lymphocytes) of disease state samples,Accordingly, the ability to analyze and monitor such disease statesamples by traditional enrichment/staining techniques is severelylimited. Where such analysis are to be performed, the more traditionalcentrifugation/density gradient enrichment protocols are preferred,since they are somewhat less disruptive of the lymphocytes than theeffects of the lytic reagents.

The enhancement in accuracy and sensitivity observed by Caldwell in his"no-wash" procedure represents a step in the appropriate direction,however, further improvement is obviously necessary, particularly whererelative cell population determinations are critical to an accurateanalysis of the patients conditions. As is evident from the abovediscussion, none of the prior art techniques, even that described byCaldwell, provide a complete solution to this problem, since virtuallyevery procedure, even those utilizing lytic reagents, occasion astatistically significant loss or destruction in one or more of thesub-populations of leukocytes; and at least 1 hour total preparationtime. This loss is statistically more significant in dealing with anaged sample (i.e. a sample essentially totally deficient in endogenousnutrient) and disease state samples because of the relatively highersensitivity of the nutritionally deprived cells, and cells indicative ofthe disease state of lysis by physical and chemical trauma.

SUMMARY OF THE INVENTION

The objective of this invention are to provide a method and system forthe conservation of the cellular analytes of content of a sample toinsure that the analysis thereof accurately reflect both the number ofthe cellular analytes of interest in the sample and; where appropriate,the relative concentration of such analytes to other cells of thesample. Thus, this invention is concerned with preparation of the sampleso as to insure that virtually all diagnostically relevant components ofthe sample are conserved. In the analysis of whole blood, thediagnositcally relevant component of particular interest is thenon-erythrocyte fraction. This invention provides the ability toeffectively isolate and yet effectively conserve the non-erythrocytefraction without the washing and multiple transfer steps traditionallyassociated with that process. In the preferred embodiments of thisinvention, a whole blood sample is placed in an incubation chamber. Theincubation chamber of choice is the same type of vessel which is used inthe instrument selected for analysis of the sample. This process ofpreparation of the sample departs from the norm in that virtually allcontact of the sample with reagents is performed in the incubationchamber and the intimate interaction of the sample and such reagents isassured by asymmetric vortexing of the content of the chamber at theappropriate intervals, frequency and for the appropriate duration. Byprecisely controlling the reagent additions, reaction environment andthe physical forces acting upon the sample, it is now possible toprepare a whole blood sample, (even aged or disease state samples)without washing or statistically significant in any of thediagnostically significant non-erythrocyte blood components.

More specifically, this method and system provides a conservative methodand system in which the mixing dynamics and reagents effect rapid andessentially complete hemolysis of the erythrocyte fraction of a wholeblood sample while maintaining the non-erythrocyte fraction of thesample in its essentially native physiological state. This method andsystem are, thus, compatible with the selective staining of one or moresub-populations of the leukocyte fraction, either prior to, orconcurrent with, hemolysis of the erythrocytes. Subsequent to suchstaining, the sample can be subjected to analysis by semi-automated orautomated techniques (i.e. flow cytometry) for identification andquantification of the cell populations of interest. This process ofenrichment and standing of the leukocyte fraction of the sample can beperformed within about sixty (60) seconds or less, provides improvedclustering of the various sub-populations of the leukocytes within thesampler; and, is conservative of the integrity and of the total numberof leukocytes present in the sample.

In one of the preferred embodiments of this invention, the whole bloodsample and stain are initially combined in an incubation/mixing chamber.A 12×75 mm test tube, of the type commonly used with the EPICS brandflow cytometer (available from Coulter Electronics), is generallysuitable for this purpose. The test tube and its contents can then beaggitated on a suitable mixing device (i.e. Q-PREP™ mixer also availablefrom Coulter Electronics). This mixer has the capability for continuousand controlled mixing of the fluid contents of the test tube and theautomated introduction of additional reagents at the appropriatepre-programmed interval. Initially, an immunological stain can becontacted with the whole blood sample in the incubation chamber,accompanied by gentle, asymmetric vortex mixing. After a suitableincubation period, generally less than sixty (60) seconds, adifferentiation effective amount of a novel lytic reagent is contactedwith the sample in the chamber and, thereafter its lytic activitysubstantially retarded by the introduction of a suitable quench. Theentire process of staining, lysing of the erythrocytes and quenching ofthe activity of the lytic reagent is performed without prior physicalisolation of the cells from one another, without transfer of the cellsof the sample from one vessel to another and without the need forremoval of unreacted stain and/or unconsumed reagents from theincubation chamber prior to analysis or measurement of the sample. Thestained leukocyte fraction can now be subjected to further analysis oninstrumentation designed for that purpose (i.e. EPICS Model C or PROFILEflow cytometer). The analysis of the sample on such instrumentationenables the generation of a histogram with well-defined clustering ofthe stained leukocyte fraction of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scattergram of the stain sample of Example I.

FIG. 2 is a scattergram of the stain sample of Example II.

FIG. 3 is a scattergram of the stain sample of Example III.

FIG. 4 is a scattergram of the stain sample of Example IV.

FIG. 5 is a scattergram of the stain sample of Example V.

FIG. 6 is a scattergram of the strain sample of Example VI.

DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

As indicated above, in somewhat abbreviated fashion, the method andsystem of this invention are unique in a number of significant respects:(a) the ability to sequentially or concurrently rapidly stain and enrichthe non-erythrocyte fraction of a whole blood sample; (b) theconservation of the morphology and total number of leukocytes originallypresent in the sample; (c) the preservation of the immunochemicalinteraction between the stain and the characteristic cellular componentsof the cell population of interest; (d) the preservation of thestained/enriched sample for up to twenty-four (24) hours prior toanalysis without material alteration of the characteristics size and/orshape of the subpopulation of interest and, (e) the compatibility of thereagents utilized in the staining sequence with the reagents used inboth lytic sequence and quenching sequence so as to eliminate theneccessity of washing of the sample between each such processing step.

Preliminary to entering into a detailed discussion of the method andsystem of this invention, it will be helpful to initially define anumber of terms and phrases.

The terms "stain" and "staining" as used herein are intended asdescriptive of a process for identification of a specific cellpopulation of interest so as to permit its differentiation from othercells which are also generally present in a sample undergoing analysis.

The term "conservative" as used herein is intended as descriptive ofboth the conditions and manipulative steps of the method of thisinvention which preserves not only the total number of leukocytesoriginally present in the sample, but also their gross morphology andtheir immunochemical reactivity. Such conservation is achieved withoutprior fixation of the leukocyte population, but can include fixationsubsequent to such preparation.

The term "nutative" and phrase "nutative motion" as used herein isintended as descriptive of an asymmetric or eccentric mixing action of areaction vessel, wherein the sample and various reagents (stain, lyticreagent, quench and fixative-if any) are maintained in a mild state ofagitation. This type of movement of the reaction vessels cause the fluidcontents of the vessel to asymmetrically distribute itself within thevessel, rising up to a greater height on one side of the vessel than theother. Because of this asymmetry, the vortex which forms in the vesseldoes not behave in the traditional manner; and, fluid mixing appears tooccur as a result of an essentially circular, planar motion. Mixingequipment suitable for use in the method and system of this invention isavailable from Coulter Electronics of Hialeah, Florida under the Q-PREPbrand name.

The term "enrichment" as used herein is intended as descriptive, in thecontext of this invention, of the relative increase in ease ofidentification (i.e. "visibility") of the leukocyte population of awhole blood sample upon lysis of the erythrocyte fraction of the sample;and, in the context of the prior art, the physical separation of theleukocyte population (i.e. buffy coat) from the other constituents ofthe whole blood sample.

As noted previously in the discussion of the relevant prior art, thepreferred technique for staining of the leukocyte fraction involves animmunochemical interaction of an indicator/antibody conjugate with asurface marker which is characteristic of the cell fraction of interest.The relative avidity of the conjugate for a specific surface marker ofinterest, can vary depending upon the precise location and conformationof the epitopic site on the cell surface. Where the cell population ofinterest, or cell count, is indicative of a disease state, these cellstend to be more fragile and, thus their interaction with the stain moresensitive to physical trauma and to chemical imbalance in the sampleenvironment. Thus, the abbreviated contact with the lytic reagent(generally less than ten (10) seconds) and the subsequent restoration ofthe ionic balance of the leukocyte fraction (by the addition of thequench) is conservative of both the total numbers and of the qualitiesof the cells of interest and, thus, facilitates their subsequentidentification by immunochemical techniques. Since there is typically ahiatus between the time at which the sample is enriched and stained andits subsequent analysis, the sample is preferably contacted with afixative to preserve both the physical size and shape of the leukocytefraction of interest. Such fixations must of course be compatible withthe prior enrichement/staining procedures and be further compatible withthe analytical means selected for the determination/measurement of therelative concentration of the cell population of interest. Where theanalytical method of choice is flow cytometry, the fixative selectedmust, of course, not exhibit any native or autofluoresce at thewavelength of excitation energy of the fluorochrome label of theconjugate stain; or, at the emission wavelength of fluorochrome stain.Other requirements of such fixatives are to be more fully discussedhereinafter in the context of the discussion of the reagent system. Thepreferred fixative is paraformaldehyde.

In the practice of the method of this invention, peripheral blood isaseptically collected by standard venipuncture techniques in vacuumtubes containing an anticoagulant (i.e. EDTA or heparin). The specimenis, thereafter, transferred to an incubation/mixing chamber of the typetypically used in conjunction with the analytical/measuring device ofchoice. In the semi-automated equipment contemplated for practice ofthis method of invention (i.e. Q-PREP™ sample preparation device), thesample is then contacted with appropriate quantities of the stain, thelytic reagent and fixative. During this period of contact with thesevarious materials, the sample is maintained in a continuous state ofmild agitation. In one of the preferred embodiments of this inventionthe stain is combined with a whole blood sample prior to addition of thelytic reagent system and fixatives. The activity of the lytic reagent issubsequently substantially retarded by the addition of a separatequench. The quench is added within about ten (10) seconds after initialcontact of the sample with the lytic reagent. The staining is allowed tocontinue, with the entire process being completed within about ninety(90) seconds. The fixative (optional) is preferably added at theconclusion of the staining cycle and subsequent to the addition of thequench. In an alternative embodiment of this invention, the staining ofthe leukocyte fraction of interest can precede enrichment of the sample.In this embodiment of the invention, the stain is simply added to thewhole blood sample and allowed to interact with the cellular fraction ofinterest. Such interaction occurs, under mild agitation, forapproximately 15 to 60 seconds, prior to addition of the lytic agent,quench and fixative (optional).

The complement of chemicals, which are employed to effect both hemolysisof the erythrocyte fraction of the sample and subtle modification of theleukocyte fraction, and thereby facilitate their subsequent isolation,identification and/or analysis, are collectively referred to as the"lytic reagent system". In the context of this invention, such systemincludes a lytic reagent and a companion quench, which is formulated forsubstantially retarding the lytic activity of the lytic reagent andrestoring the ionic balance of the sample.

The preferred lytic reagent which is suitable for use in the method andsystem of this invention comprises an aqueous solution containing adifferentiation effective amount of a lytic reagent comprising a watersoluble, organic carboxylic acid having a pK value>3.0, a pH in therange of from about 2.6 to 4.0 and a counterion which does materiallyalter the ionic strength of the treated sample. The most preferred lyticreagents are selected from the group consisting of formic acid, aceticacid and their respective mixtures. In the preferred embodiments of thisinvention, these lytic reagent mixtures will comprise formic acid, asthe major functional component, with the acetic acid being present inonly minor amounts, if at all. The phrase "differentiation effectiveamount" is used through this disclosure as indicative of a concentrationof lytic reagent which is not only effective for hemolysis of theerythrocyte fraction of the sample, but also effects subtle changes inthe leukocyte fraction to facilitate their subsequent isolation,identification and/or analysis.

The lytic reagent employed in the method and system of this inventioncomprises an aqueous solution containing surprisingly low concentrationsof the lytic reagent (preferably less than 1.0% by volume). The lyticreagent is preferably formic acid, acetic acid or mixtures of formic andacetic acid in which formic acid is the predominant functionalcomponent. This aqueous solution is prepared by simple addition of thelytic reagent to deionized water. The amount of lytic reagent added tothis diluent is sufficient to prepare a solution containing from about0.05 to about 0.5% (v/v) solution. In the preferred embodiments of thisinvention, the concentration of lytic reagent will range from about 0.1to about 0.25% (v/v).

Where the lytic reagent comprises a mixture containing both formic andacetic acid, the acetic acid is preferably only present as a partialreplacement for a definitive amount of formic acid and then only at aconcentration in the range of from about 0.05 to 0.10% (v/v).

The lytic reagent system can also contain other traditional additives,to the extent their presence is not otherwise incompatible with theprimary functional components of the system (i.e. anti-microbialpreservatives, such as sodium omadine).

This lytic reagent system can be combined with a whole blood sample bysimple manual or automated addition, the lytic reagent and sampleallowed to briefly interact in a suitable incubation chamber, and theaction of the lytic reagent substantially retarded by addition of asuitable quench. The quench, to be effective in this environment, must,thus, be capable of retarding the lytic activity of the lytic reagentimmediately upon its addition to the aqueous mixture contained in theblood sample and the lytic reagent. The precise formulation of thequench can vary, depending upon the composition of the lytic reagentsystem and other fluids which may be dictated by the requirements of theanalytical measurement instrument. The quench is typically an aqueoussolution containing soluble salts which are both effective tosubstantially retard and/or substantially neutralize the lytic activityof the lytic reagent and restore the ionic balance to the sample. Thisrestoration of the ionic balance will extend the longevity of thesurviving cells and permit subsequent analysis on equipment whichrequires that the sample be electrically conductive (containelectrolytes), as for example with an EPICS C® flow cytometer.

A quench which is suitable for use in conjunction with the lytic reagentcomposition can, and usually will, contain any combination of at leasttwo of the following four ingredients: sodium chloride, sodium sulfate,sodium bicarbonate, sodium carbonate; and, in addition, sodium azide asa preservative. The effectiveness of the quench upon the lytic reagentin the context of the method and system invention is determined by itsability to rapidly reduce the lytic activity of the formic acid, aceticacid and the mixtures of formic and acetic acid. As noted above, themethod and equipment utilized in the differentiation of the leukocytesubpopulation can also place certain requirements (i.e. conductivity,pH, etc.) upon the precise formulation of the quench. More specifically,when such differentiation is performed in a focused flow apertureanalysis system, the composition and volume of quench can be critical tooptimal separation (differentiation) of the five (5) leukocytesubclasses from one another. In this type of analysis system, the ionicbalance of the quench must also be adjusted to obtain a satisfactoryconductivity match of the lysed blood sample to the sheath fluid. In thepreferred quench formulation, the major ionic species and their relativeratio in the lysed-quenched blood sample should be essentially the sameas the major ionic species and their relative ratio in the sheath fluid.The relative concentration of the functional components of the quench,which is to be used in conjunction with the lytic reagent system of thisinvention, will range from about 1 to about 3% (w/v) sodium chloride,about 0.25 to about 0.8% (w/v) sodium carbonate or bicarbonate, andabout 2 to about 5% (w/v) sodium sulfate. The precise relativequantities of ingredients of the optimum quench are generally determinedempirically; the objectives of such adjustment being to attain the pH ofthe lysed blood sample within the range of from about 6.0 to about 7.5,and a final osmolality of the stabilized lysed blood sample in the rangeof from about 300 to about 330 mOs. It has been previously observed, ina focused flow aperture system, that optimal clustering of the leukocytesubclasses is achieved by adjustment in the osmolality of the finalblood sample to about 310 mOs. The essentially complete neutralizationof the acidity of the lysed sample with an alkaline quench can becritical to a focused flow aperture analysis system. Components of thesample (i.e. fibrin and platelets) are pH sensitive and can formaggregates under acidic conditions which can potentially interfere withdifferential analysis (i.e. noise) or physically obstruct the apertureof a focused flow aperture system. These considerations with respect topH adjustment may also prove critical in other analytical/measurementenvironments.

In the preferred embodiments of this invention, the duration ofeffective contact of the lytic reagent and the blood sample (from thetime the two are combined, to the time when the quench is added), mustbe less than ten (10) seconds, and most preferably six seconds or less.The interval of reactive contact of the lytic reagent and blood sampleas specified above, presumes such reactive contact occurs at roomtemperature (˜18°-28° C.).

The stain which is used in the method and system of this inventioncomprises an indicator which has been conjugated to a protein which isspecific for binding to an epitope on the cell surface of a cellularanalyte of interest (i.e. B-lymphocytes and T-lymphocytes). Antibodiesspecific for leukocytes surface markers are available from a number ofcommercial sources. In the preferred embodiments of this invention amonoclonal antibody is the reagent of choice. Monoclonals which arespecific for surface markers on T-cell and B-cells can be readilyobtained commercially. In a number of instances these same monoclonalsantibodies which have been conjugated to an appropriate indicate(flurorchrome) and the resulting conjugates also commercially available.

A series of analysis were made in the evaluation of the method andreagent system of this invention by comparison to prior art techniquesused in enrichment and staining of a specific leukocyte population ofinterest (B- and T-cells). In each instance, the whole blood sample wascommon to each, as was the method/equipment for measurement of thespecific cell population of interest. The technique forseparation/enrichment of the leukocyte fraction followed the protocolestablished for each of the reagent systems designed for that purpose(Examples I-V). The method and reagent system of this invention isillustrated in Example VI. In each of these Examples, the analysis ofthe enriched sample was conducted on an EPICS Model C flow cytometer,(Coulter Electronics, Hialeah, Fla.) following the standard operatingprotocol for this instrument. The results of each analysis is depictedin a series of scattergrams, FIGS. 1, 2, 3, 4, 5 and 6; with FIGS. 1-5representing the prior art and FIG. 6 the method/reagent system of thisinvention.

EXAMPLE I

The separation media and method used in the preparation of the samplefor this Example involved separation/enrichment of the leukocytefraction by traditional lysis techniques, followed, thereafter bystaining with a fluorochrome labeled antibodies which are specific for Band T cells, respectively; Ortho Diagnostic System, procedure forpreparation and staining of mononuclear cells for analysis on ORTHOSPECTRUM III Laser Flow Cytometry System, CYTOFLUOROGRAF™. Followingpreparation of the sample, the relative concentration of B and T cellsis determined in an EPICS Model C flow cytometer. FIG. 1A represents ascattergram of the leukocyte sub-populations of whole blood sample froma healthy donor. FIG. 1B represents the relative B-cell concentration(12.20%) FIG. 1C represents the relative T-cell concentration (83.30%).

EXAMPLE II

Sample preparation involved lysis of erythrocytes with a detergent inaccordance with the procedures described for the reagent system marketedunder the Immunolyse label by Coulter Immunology. Sample analysis isperformed in the same manner as for the sample of FIG. 1 FIG. 2Arepresents a scattergram of the leukocytes sub-population of a wholeblood sample from the same healthy donor as depicted in the scattergramof FIG. 1. FIG. 2B represents the relative concentration of B-cells inthe sample. FIG. 2C represents the relative concentration of T-cells inthe sample. Note the variations in results in comparison of reportedrelative concentration of B cells and T cells with the values reportedfor the analysis conducted in accordance with FIG. 1.

EXAMPLE III

Sample preparation involved lysis of erythrocytes with diethylene glycolin accordance with the procedures described for the reagent systemmarketed under the FACS label by Becton-Dickinson. FIG. 3A represents ascattergram of this leukocyte sub-population of a whole blood samplefrom the same healthy donor as in FIGS. 1A and 2A. FIG. 3B is anenhanced scattergram representing the relative concentration of theB-cells in sample (19.04%). FIG. 3C is an enhanced scattergramrepresenting the relative concentration of the T-cells in the sample(69.17%). Note the lack of correlation of the results with thescattergram of FIGS. 1 and 2. Moreover, the relative concentration B toT cells has also shifted dramatically, indicating that the samplepreparation (lysis of RBC's with diethylene glycol) is unduly harsh uponT cells.

EXAMPLE IV

Sample preparation involved density gradient separation of primarilylymphocyte and monocyte sub-populations. This procedure is acknowledgedto be highly techician dependant and reportedly prone to substantialcell losses. FIG. 4A represents a scattergram of the leukocyte subpopulations of a whole blood sample from the same healthy donor as inFIGS. 1A, 2A and 3A. FIG. 4B is an enhanced scattergram representing therelative concentration of the B-cells in such sample (4.64%). FIG. 4C isan enhanced scattergram representing the relative concentration of theT-cell in such sample (88.55%). Note the lack of correlation of theresults with the scattergrams of FIGS. 1, 2 and 3. Moreover, therelative concentration of B cells to T cells has also dramaticallyshifted indicating that repeated washing and physical manipulation ismore traumatic to B cells than T cells.

EXAMPLE V

The procedures of Example IV is repeated except that the unreacted stainis allowed to remain in the incubation chamber and the analysisconducted as specified per Caldwell et al "no wash". The scattergram ofFIG. 5A represents the leukocyte subpopulation which are capable ofdifferentiation. FIG. 5B is an enhanced scattergram representing therelative concentration of the B-cells (8.14%). FIG. 5C is an enhancedscattergram representing the relative concentration of the T-cells(87.75%). Note the increased relative concentration of the B-cellpopulation, thus, confirming the Caldwell et al observation regardingthe advantages of elimination of the wash step following staining of thesample.

EXAMPLE VI

Sample preparation involved rapid (less than 90 seconds total elapsedtime) staining/lysis/quench/fixation of the sample. Sample preparationinvolved the automated dispensing/mixing of the following reagents, inprescribed sequence, with the a whole blood sample from the same donoras in Examples I-V.

    ______________________________________                                        I. Materials                                                                  ______________________________________                                        1. Lyse formulation:                                                                        1.2 mL formic acid                                                            0.2 mL sodium omadine (40)                                                    998.6 mL deionized water                                        2. Quench Formulation:                                                                      31.30 grams soduim sulfate                                                    14.50 grams sodium chloride                                                    6.00 grams dodium carbonate                                                  1 liter deionized water                                         3. Fixative:   6.05 grams TRIS (HCL) buffer                                                 10.00 grams paraformaldehyde                                                  00.05 mL sodium hydroxide (50%)                                               1 liter deionized water                                         4. Stain:     Becton-Dickinson                                                              Immunocytometry Systems                                                       G1-FITC                                                                       HLE-FITC                                                                      Coulter Immunology Division                                                      MIG-FITC  TY-RD1                                                              MIG-RD1   T1-FITC                                                             T8-FITC   B1-FITC                                            ______________________________________                                    

The device (Q-PREP™ available from Coulter Electronics, Hialeah,Florida) used in the programmed addition of the stain, lytic reagentsystem and fixative delivered a pre-set amount of each of these reagentsat the appropriate interval during mild agitation of a 25×75 mm testtube operating on the following cycle:

(a) addition of fluorochrome labeled antibody to sample

(b) mild agitation of sample stain for 60 seconds

(c) addition of lytic reagent to stained sample

(d) agitate for 6 seconds

(e) addition of quench

(f) agitate for 10 seconds

(g) addition of fixative

(h) place test tube in flow cytometer and analyze for relativepopulation of B-cells and T cells.

FIG. 6A is a scattergram of the leukocyte sub-population of a wholeblood sample from the same healthy donor as in FIGS. 1A, 2A, 3A, 4A and5A. FIG. 6B is an enhanced scattergram representing the relativeconcentration of the B-cells in such sample (19.79%). FIG. 6C is anenhanced scattergram representing the relative concentration of T-cellsin such sample (76.97%). Note the relating high concentration of B-cellsindicating that the method and reagent system of this invention areeffective in the identification of the relevant sub-population ofinterest and that this technique is efficacious with minimal technicianinvolvement.

What is claimed is:
 1. In a method for the preparation and analysis of awhole blood sample by photooptical measurement techniques, whereinsample preparation includes enrichment of a non-erythrocyte cellularfraction of the sample by selective stromatolysis of sample erythrocyteswith a lytic reagent and labelling of one or more sub-populations of thenon-erythrocyte cellular fraction with an indicator labelled bindingmaterial which is specific for a characteristic cellular component of asub-population of the non-erythrocyte cellular fraction, wherein theimprovement comprises:(a) enriching the non-erythrocyte cellularfraction of the whole blood sample and labelling one or moresub-populations of the non-erythrocyte cellular fraction by contactingthe sample with an indicator labelled binding material and a lyticreagent system comprising an organic carboxylic acid with a pK greaterthan 3.0 in a reaction vessel with gentle asymmetric vortex mixing; (b)transferring an aliquot of selectively stromatolysed sample to aphotooptical analyzer without separation of labelled sub-populations ofthe non-erythrocyte cellular fraction from unconsumed lytic reagent ofunbound indicator labelled binding material; and (c) subjecting saidaliquot to photooptical analysis.
 2. The method of claim 1, whereinsample preparation involves stromatolysis of the erythrocyte fraction ofthe sample with an effective amount of an acidic lytic reagent, having apH in the range of from about 2.6 to about 4.0, quenching the activityof said lytic reagent with a companion reagent which is effective inarresting the lytic activity of the lytic reagent and restoring theIonic Balance of the sample.
 3. The method of claim 2 wherein samplepreparation involves contacting the sample with reagents concurrent withgentle asymmetric vortex mixing so as to maintain sample particulates atan essentially homogeneous concentration throughout the sample, saidpreparation requiring less than 90 seconds.
 4. The method of claim 2,wherein the sample is contacted with an indicator labelled bindingmaterial before the sample is contacted with the lytic reagent.
 5. Themethod of claim 2, wherein the sample is contacted with an indicatorlabelled binding material concurrent with the lytic reagent.
 6. Themethod of claim 2, wherein the sample is contacted with an indicatorlabelled binding material after the sample is contacted with the lyticreagent.
 7. The method of claim 2, wherein the sample is contacted witha fixative subsequent to staining with the indicator labelled bindingmaterial.
 8. The method of claim 2, wherein the sample comprises an agedwhole blood specimen wherein the endogenous nutrients of the sample havebeen essentially completely depleted and the cells are fragile.
 9. Themethod of claim 2, wherein the sample comprises a whole blood specimenfrom a patient suspected of suffering from a disease which involveschanges in at least one of non-erythrocyte cellular morphology andpopulation.
 10. The method of claim 1, wherein the lytic reagent is anorganic acid selected from the group consisting of formic acid, aceticacid, and mixtures thereof.
 11. The method of claim 1 in which saidselective stromatolysis is accomplished in approximately 10 seconds. 12.The method of claim 11 in which labelling of said non-erythrocytecellular fraction is effected in about 15 to 60 seconds, prior to saidstromatolysis.